ANTI-TISSUE FACTOR MONOCLONAL ANTIBODY

- NATIONAL CANCER CENTER

Monoclonal antibodies against human and mouse tissue factor, and fragments thereof, are disclosed, as well as pharmaceutical compositions and compositions for drug delivery containing the same. Therapeutic methods using the same are also disclosed.

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Description
TECHNICAL FIELD

The present invention relates to anti-tissue factor monoclonal antibodies and to pharmaceutical compositions utilizing the antibodies.

BACKGROUND ART

This description encompasses the contents described in the specification and/or the drawings of Japanese Patent Application No. 2014-18586, from which the present application claims priority. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

In general, when a drug is systemically administered orally or by intravenous injection, the drug is supplied to not only a focus serving as a target of the drug administration but also to normal tissue. As a result, side effects of the drug administration are observed and in some cases the treatment method needs to be changed or stopped. In view of this, for the purpose of reducing side effects, drugs called molecularly targeted drugs have been developed, which have the ability to specifically bind to a molecular marker, such as a receptor, a ligand, or an enzyme, which is unique to the target of the drug administration (for example, Patent Literature 1).

Meanwhile, tissue factor (hereinafter sometimes referred to as “TF”) is an initiator of extrinsic coagulation, and its production is promoted by vascular injury or the like. Expression of TF is local and transient in a normal response. However, it is known that, in many solid cancers, such as pancreatic cancer and stomach cancer, the expression of TF is constitutively enhanced at cell surfaces of, for example, cancer cells, vascular endothelial cells, monocytes, and macrophages in tumor tissues.

CITATION LIST Patent Literature

  • Patent Literature 1: US 2012/0039989 A1

SUMMARY OF THE INVENTION Problem(s) to be Solved by the Invention

An object of the present invention is to provide a novel antibody against TF. In addition, another object of the present invention is to provide a pharmaceutical composition utilizing the antibody as a target-binding factor.

Means for Solving the Problem(s)

The inventors of the present application have found novel anti-TF monoclonal antibodies having an ability to be internalized by a cell, and further have conceived that a drug can be delivered with high selectivity to a cell expressing TF at its surface by using the antibodies as a target-binding factor, thereby completing the present invention.

That is, according to the present invention, the following monoclonal antibodies, which bind to tissue factor, are provided.

An anti-human tissue factor monoclonal antibody including a heavy chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 3, 4, and 5, respectively, and a light chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 6, 7, and 8, respectively;

an anti-human tissue factor monoclonal antibody including a heavy chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 11, 12, and 13, respectively, and a light chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 14, 15, and 16, respectively; or

an anti-mouse tissue factor monoclonal antibody including a heavy chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 19, 20, and 21, respectively, and a light chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 22, 23, and 24, respectively.

According to another aspect of the present invention, monoclonal antibodies are provided, which bind to the same epitope as an epitope of tissue factor to which an above-mentioned monoclonal antibody binds.

According to yet another aspect of the present invention, antibody fragments are provided that include part of an above-mentioned monoclonal antibody, the antibody fragments being capable of binding to tissue factor.

According to yet another aspect of the present invention, pharmaceutical compositions are provided that include: an above-mentioned monoclonal antibody or an above-mentioned antibody fragment as a target-binding factor; and a drug.

According to yet another aspect of the present invention, compositions for drug delivery are provided that include an above-mentioned monoclonal antibody or an above-mentioned antibody fragment as a target-binding factor.

Effects of the Invention

Monoclonal antibodies of the present invention can recognize a cell expressing TF and can have an ability to be internalized by the cell. Accordingly, by using the antibodies as a target-binding factor, a drug can be efficiently delivered to the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are micrographs that show the results of an internalization assay.

FIG. 2 is a graph that shows the results of an anticoagulant activity evaluation.

FIG. 3 is a graph that shows the results of a cytocidal effect confirmation test.

FIG. 4 is a graph that shows changes in tumor volume in an antitumor effect confirmation test.

FIG. 5 is a graph that shows changes in body weight in the antitumor effect confirmation test.

FIG. 6 is a micrograph that shows the result of an internalization assay.

FIG. 7 is a graph that shows the ratios of mRNA expression amounts of TF to mRNA expression amounts of GAPDH in mouse B16 melanoma cells and TF forced-expression cells thereof.

FIG. 8 is a histogram obtained by FACS analysis.

 MODES FOR CARRYING OUT THE INVENTION A. Monoclonal Antibody

According to the present invention, monoclonal antibodies that bind to TF are provided. Typically, monoclonal antibodies of the present invention are capable of binding to TF and have an ability to be internalized by a cell expressing TF. TF is blood coagulation factor III, and is expressed at the cell surface as a transmembrane glycoprotein. In the present invention, TF is preferably human TF (hTF). The full-length amino acid sequence of hTF is already known under GenBank ACCESSION_AAA61152 (SEQ ID NO: 1). In the present invention, a monoclonal antibody against mouse TF (mTF) is also provided. The utilization of the anti-mTF monoclonal antibody as a target-binding factor for a drug can be effective in testing or research using mice. The full-length amino acid sequence of mTF is already known under GenBank ACCESSION_AAA63400 (SEQ ID NO: 2).

Herein, internalization means a phenomenon in which an antibody forms an immunocomplex with an antigen at the cell surface and is then taken up into the cell. Whether or not the anti-TF monoclonal antibody has the ability to be internalized may be determined by, for example: a method involving bringing an antibody, which has a labeling substance bound thereto, into contact with a cell expressing TF at its surface, and confirming whether or not the labeling substance has been transferred into the cell; or a method involving bringing an antibody, which has a cytotoxic substance bound thereto, into contact with a cell expressing TF at its surface, and confirming whether or not the contact induces cell death or cell growth inhibition. More specifically, the presence or absence of the ability of an antibody to be internalized may be confirmed by the internalization assay described in Examples.

Any appropriate cell may be used as the cell expressing TF at its surface, and examples thereof include cells in tumor tissues. The expression of TF in a normal tissue is normally a local and transient expression, whereas the expression of TF is constitutively enhanced at cell surfaces of, for example, cancer cells, vascular endothelial cells, monocytes, and macrophages in tumor tissues. Specific examples of the cancer cells include pancreatic cancer cells and stomach cancer cells.

Herein, “monoclonal antibody” refers to antibodies produced by antibody-producing cells that are monoclonal. Monoclonal antibodies have uniform primary structures and recognize the same epitope. Monoclonal antibodies of the present invention have a basic structure formed of a tetramer in which two identical heavy chains and two identical light chains are bound by disulfide bonds. Anti-TF monoclonal antibodies of the present invention may be any isotype of IgG, IgA, IgM, IgD, or IgE. Of those, IgG is preferred.

An epitope that anti-TF monoclonal antibodies of the present invention recognizes is preferably present in the extracellular domain of TF.

The dissociation constant (KD) of anti-TF monoclonal antibodies of the present invention for TF reaches, for example, 5×10−9 M or less, even 1×10−9 M or less, particularly 2×10−10 M or less. The dissociation constant may be measured, for example, using a surface plasmon resonance method.

Anti-IF monoclonal antibodies of the present invention may or may not exhibit anticoagulant activity. The presence or absence of anticoagulant activity or its degree may be determined based on prothrombin time (PT). An anti-TF monoclonal antibody exhibiting no anticoagulant activity or low anticoagulant activity is hardly captured by a blood clot or the like, and hence its utilization as a target-binding factor for a drug can improve the deliverability of the drug to a target site, with the result that the efficacy of the drug can be suitably exhibited. The prolonged coagulation time ratio (ratio relative to PBS) of anti-TF monoclonal antibodies of the present invention, in the state that an antigen-antibody complex has been formed, is preferably 3 or less, more preferably 2 or less, still more preferably from 1 to 1.5. The prolonged coagulation time ratio, in the state that an antigen-antibody complex has been formed, may be determined by the method described in Examples.

[A-1. Anti-hTF Monoclonal Antibodies]

In a first embodiment, an anti-hTF monoclonal antibody of the present invention includes a heavy chain variable region having complementarity determining regions (CDR) 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 3, 4, and 5, respectively, and a light chain variable region having CDR1, CDR2, and CDR3 containing the amino acid sequences set forth in SEQ ID NOS: 6, 7, and 8, respectively. A preferred specific example thereof may be an anti-hTF monoclonal antibody including a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 10.

In a second embodiment, an anti-hTF monoclonal antibody of the present invention includes a heavy chain variable region having CDR1, CDR2, and CDR3 containing the amino acid sequences set forth in SEQ ID NOS: 11, 12, and 13, respectively, and a light chain variable region having CDR1, CDR2, and CDR3 containing the amino acid sequences set forth in SEQ ID NOS: 14, 15, and 16, respectively. A preferred specific example thereof may be an anti-hTF monoclonal antibody including a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 18.

Variants of each monoclonal antibody exemplified in the first or second embodiment may also be encompassed in anti-hTF monoclonal antibodies of the present invention. Examples of the variants are monoclonal antibodies, in which the heavy chain variable region and/or the light chain variable region contain(s) one or several (for example, one to ten, preferably one to five) amino acid substitutions, insertions, additions, and/or deletions. Such variants can also suitably bind to hTF and have the ability to be internalized by a cell expressing hTF.

Specific examples of variants of the monoclonal antibodies are monoclonal antibodies including a heavy chain variable region containing an amino acid sequence which is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more identical to the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region containing an amino acid sequence which is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more identical to the amino acid sequence set forth in SEQ ID NO: 10. In addition, other specific examples of the variants are monoclonal antibodies including a heavy chain variable region containing an amino acid sequence which is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more identical to the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region containing an amino acid sequence which is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more identical to the amino acid sequence set forth in SEQ ID NO: 18. It is preferred that any such variant be capable of binding to hTF and have the ability to be internalized by a cell expressing hTF.

Variants of the monoclonal antibodies may contain, in at least one of the CDRs of the heavy chain variable region and/or the light chain variable region of its corresponding monoclonal antibody, one or several, for example, one, two, or three, preferably one or two, more preferably one amino acid substitution, insertion, addition, and/or deletion. Each CDR of the variant has a homology of preferably from 90% to 100% to each CDR of its corresponding monoclonal antibody, and the homology is more preferably from 95% to 100%, still more preferably from 98% to 100%, most preferably 100%. In addition, the entire CDR1 to CDR3 of the heavy chain and the light chain of the variants have a homology of preferably from 90% to 100% to the entire CDR1 to CDR3 of the heavy chain and the light chain of its corresponding monoclonal antibody, and the homology is more preferably from 95% to 100%, still more preferably from 98% to 100%, most preferably 100%.

In a third embodiment, anti-hTF monoclonal antibodies of the present invention may be monoclonal antibodies that bind to the same epitope as an epitope of hTF to which the monoclonal antibodies exemplified in the first or second embodiment binds. Antibodies that bind to the same epitope may be obtained by a known method such as a competitive ELISA method. In a competitive ELISA method, for example, if the antibody serving as the test subject decreases the binding activity of a control antibody (that is, the monoclonal antibody exemplified in the first or second embodiment) by 30% or more, preferably 40% or more, more preferably 50% or more, as compared to the binding activity of the control antibody in the absence of the antibody serving as the test subject, the antibody serving as the test subject may be said to be an antibody that binds to substantially the same epitope as the control antibody. It is preferred that the antibody that binds to the same epitope be capable of binding to hTF and have the ability to be internalized by a cell expressing hTF. It should be noted that, in such embodiments, the antibody that binds to the same epitope may be a variant of the monoclonal antibodies exemplified in the first or second embodiment.

Anti-hTF monoclonal antibodies of the present invention described above may be a human chimeric antibody or a humanized antibody.

“Human chimeric antibody” refers to an antibody in which a variable region of an antibody of non-human mammalian origin and a constant region of an antibody of human origin are linked to each other. Accordingly, human chimeric antibodies of the present invention may be a chimeric antibody obtained by linking the heavy chain variable region and the light chain variable region of a monoclonal antibody exemplified in the first, second, or third embodiment to a human heavy chain constant region and a human light chain constant region, respectively.

Specifically, an example of a human chimeric antibody of the present invention is a chimeric antibody in which a heavy chain variable region containing the amino acid sequences set forth in SEQ ID NOS: 3, 4, and 5 as heavy chain CDR1, CDR2, and CDR3, respectively, and a light chain variable region containing the amino acid sequences set forth in SEQ ID NOS: 6, 7 and 8 as light chain CDR1, CDR2, and CDR3, respectively are linked to a human heavy chain constant region and a human light chain constant region, respectively. A specific example of such a chimeric antibody is a chimeric antibody in which a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 10 are linked to a human heavy chain constant region and a human light chain constant region, respectively.

Another example of the human chimeric antibody of the present invention is a chimeric antibody in which a heavy chain variable region containing the amino acid sequences set forth in SEQ ID NOS: 11, 12, and 13 as heavy chain CDR1, CDR2, and CDR3, respectively, and a light chain variable region containing the amino acid sequences set forth in SEQ ID NOS: 14, 15, and 16 as light chain CDR1, CDR2, and CDR3, respectively are linked to a human heavy chain constant region and a human light chain constant region, respectively. A specific example of such a chimeric antibody is a chimeric antibody in which a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 17 and alight chain variable region containing the amino acid sequence set forth in SEQ ID NO: 18 are linked to a human heavy chain constant region and a human light chain constant region, respectively.

The heavy chain constant region of the human chimeric antibody only needs to be one belonging to a human immunoglobulin (hereinafter described as hIg), and preferably belongs to the hIgG class. Similarly, the light chain constant region of the human chimeric antibody only needs to be one belonging to hIg, and may be belong to either one of the κ class and the λ class.

“Humanized antibody” refers to an antibody in which CDRs of an antibody of non-human mammalian origin are grafted at the appropriate location in a variable region of an antibody of human origin. Accordingly, humanized antibody of the present invention may be a human antibody which has the CDR1 to CDR3 of the heavy chain and the light chain of a monoclonal antibody exemplified in the first, second, or third embodiment, as the CDR1 to CDR3 of the heavy chain and the light chain, and in which the other regions are derived from a human antibody.

Specific examples of humanized antibodies of the present invention include: a humanized antibody in which CDR1, CDR2, and CDR3 of the heavy chain have the amino acid sequences set forth in SEQ ID NOS: 3, 4, and 5, respectively, CDR1, CDR2, and CDR3 of the light chain have the amino acid sequences set forth in SEQ ID NOS: 6, 7, and 8, respectively, and the other regions are derived from a human antibody; and a humanized antibody in which CDR1, CDR2, and CDR3 of the heavy chain have the amino acid sequences set forth in SEQ ID NOS: 11, 12, and 13, respectively, CDR1, CDR2, and CDR3 of the light chain have the amino acid sequences set forth in SEQ ID NOS: 14, 15, and 16, respectively, and the other regions are derived from a human antibody.

The heavy chain of the humanized antibody only needs to be one belonging to hIg, and preferably belongs to the hIgG class. Similarly, the light chain of the humanized antibody only needs to be one belonging to hIg, and may belong to either one of the κ class and the λ class.

[A-2. Anti-mTF Monoclonal Antibodies]

In one embodiment, an anti-mTF monoclonal antibody of the present invention includes a heavy chain variable region having CDR1, CDR2, and CDR3 containing the amino acid sequences set forth in SEQ ID NOS: 19, 20, and 21, respectively, and a light chain variable region having CDR1, CDR2, and CDR3 containing the amino acid sequences set forth in SEQ ID NOS: 22, 23, and 24, respectively. A preferred specific example thereof may be an anti-mTF monoclonal antibody including a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 26.

Variants of the monoclonal antibodies exemplified above may also be encompassed in anti-mTF monoclonal antibodies of the present invention. Examples of variants are monoclonal antibodies in which the heavy chain variable region and/or the light chain variable region contain(s) one or several (for example, one to ten, preferably one to five) amino acid substitutions, insertions, additions, and/or deletions. Such variants can also suitably bind to mTF and have the ability to be internalized by a cell expressing mTF.

Specific examples of variants of the monoclonal antibodies are monoclonal antibodies including a heavy chain variable region containing an amino acid sequence which is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more identical to the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region containing an amino acid sequence which is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more identical to the amino acid sequence set forth in SEQ ID NO: 26. It is preferred that such variants be capable of binding to mTF and have the ability to be internalized by a cell expressing mTF.

Variants of the monoclonal antibodies may contain, in at least one of the CDRs of the heavy chain variable region and/or the light chain variable region of its corresponding monoclonal antibody, one or several, for example, one, two, or three, preferably one or two, more preferably one amino acid substitution, insertion, addition, and/or deletion. Each CDR of the variants has a homology of preferably from 90% to 100% to each CDR of its corresponding monoclonal antibody, and the homology is more preferably from 95% to 100%, still more preferably from 98% to 100%, most preferably 100%. In addition, the entire CDR1 to CDR3 of the heavy chain and the light chain of the variant have a homology of preferably from 90% to 100% to the entire CDR1 to CDR3 of the heavy chain and the light chain of its corresponding monoclonal antibody, and the homology is more preferably from 95% to 100%, still more preferably from 98% to 100%, most preferably 100%.

In another embodiment, anti-mTF monoclonal antibodies of the present invention may be monoclonal antibodies that bind to the same epitope as the epitope of mTF to which the monoclonal antibody exemplified above binds. It is preferred that antibodies that binds to the same epitope be capable of binding to mTF and have the ability to be internalized by a cell expressing mTF. In such embodiments, the antibodies that bind to the same epitope may be variants of the monoclonal antibody exemplified above. It should be noted that a method of obtaining antibodies that bind to the same epitope is described above.

B. Production Method for Monoclonal Antibodies B-1. Production of Monoclonal Antibodies Using Hybridomas

Monoclonal antibodies of the present invention may be obtained by, for example, preparing hybridomas through cell fusion between antibody-producing cells obtained from an animal immunized with an antigen and myeloma cells, selecting, from the resultant hybridomas, hybridomas which produce an antibody of interest, and allowing the selected hybridomas to produce the antibody.

B-1-1. Preparation of Antigen

As the antigen to be used in the immunization of the animal, for example, TF (full-length TF) or a partial peptide thereof, or a cell expressing TF at its surface may be used. hTF may be obtained by, for example, purifying hTF derived from a human placenta according to the method disclosed in JP 09-302000 A or the like. In addition, for example, TF or a partial peptide thereof may be obtained by a genetic engineering method or a chemical synthesis method. A partial peptide may be used by being bound to any appropriate carrier protein as necessary. It should be noted that the mRNA sequence of hTF is known under GenBank NM_001993.4 (SEQ ID NO: 27). In addition, the mRNA sequence of mTF is known under GenBank M57896.1 (SEQ ID NO: 28).

B-1-2. Preparation of Antibody-Producing Cells

The antigen obtained as described above is mixed with any appropriate adjuvant, and is administered to a non-human mammal, such as a mouse, a rat, a horse, a monkey, a rabbit, a goat, or a sheep, to immunize the non-human mammal. The antibody titer of the immunized animal against the antigen is measured, and an animal having a high antibody titer is subjected to final immunization. Several days after the day of the final immunization, antibody-producing cells, such as spleen cells or lymph node cells, are collected. Details of a method for the immunization and a method of collecting the antibody-producing cells are well known to persons skilled in the art, and hence a detailed description thereof is omitted. The antibody titer may be measured by, for example, an enzyme immunoassay (EIA), such as an ELISA method, or a radioimmunoassay (RIA), with blood collected from the animal.

B-1-3. Cell Fusion

As the myeloma cells to be fused with the antibody-producing cells, any appropriate cell line which is derived from an animal, such as a mouse or a rat, and which is generally available to persons skilled in the art may be used. It is preferred to use myeloma cells having drug resistance and having the following properties: being unable to survive in a selection medium (such as a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium)) in an unfused state and being able to survive therein only in a fused state. The cell fusion may be performed using any appropriate method, such as a PEG method or an electrofusion method. Then, after the cell fusion treatment the cells are suspended and diluted in a selection medium (such as HAT medium), and cultured in wells of a culture plate.

B-1-4. Screening and Cloning of Hybridomas

Cells which have formed colonies as a result of culturing after the cell fusion are selected as hybridomas. Then, the selected hybridomas are, for example, cultured in a microtiter plate, and the resultant culture supernatant is collected and measured for reactivity to the antigen. The reactivity to the antigen may be measured by EIA, RIA, or the like. Hybridomas showing reactivity to the antigen as a result of the measurement are selected, and monoclonal antibody-producing hybridomas are isolated by a limiting dilution method or the like.

B-1-5. Preparation of Monoclonal Antibodies from Hybridomas

Monoclonal antibodies may be prepared by, for example: a method involving culturing the hybridomas in any appropriate medium, and purifying the monoclonal antibody from the resultant culture supernatant; or a method involving injecting the hybridomas into the abdominal cavity of a non-human mammal, such as a mouse or a rat, to culture the hybridomas in peritoneal fluid, and purifying the monoclonal antibody from the resultant peritoneal fluid. The antibodies may be purified by using, for example, an ammonium sulfate precipitation method, a gel-filtration chromatography method, an ion-exchange chromatography method, and an affinity column chromatography method, such as an anti-immunoglobulin column or a protein A column in combination as necessary.

B-2. Production of Monoclonal Antibodies Using Genetic Engineering Techniques

Monoclonal antibodies of the present invention may be produced by, for example, genetic engineering techniques through the utilization of an antibody gene cloned from antibody-producing cells, such as the hybridomas.

B-2-1. Cloning of Gene Encoding Variable Region

Total mRNA is extracted from hybridomas that produce the antibody of interest, and cDNA encoding an antibody variable region is synthesized from the resultant total mRNA with reverse transcriptase by using sequences common to antibody genes as primers. The synthesis and amplification of the cDNA may be performed using, for example, a 5′-RACE method, and at that time, any appropriate restriction enzyme site may be introduced at both ends of the cDNA. A DNA fragment of interest is purified from the resultant PCR product, linked to vector DNA, and introduced into Escherichia coli or the like, to thereby prepare a desired recombinant vector. Then, the base sequence of the antibody gene of interest is confirmed by a known method, such as a deoxy method.

B-2-2. Introduction of Antibody Gene into Host Cell

The DNA encoding the variable region cloned as described above is linked to DNA encoding a desired antibody constant region, and the resultant is incorporated into an expression vector. As an alternative, the DNA encoding the variable region may be incorporated into an expression vector containing DNA encoding the desired constant region. The thus-obtained expression vector may be introduced into any appropriate host cell, to thereby express the antibody. In this case, the heavy chain and the light chain may be separately incorporated into expression vectors, followed by simultaneous introduction of these two expression vectors into the same host cell, or DNA encoding the heavy chain or the light chain may be incorporated into a single expression vector to be introduced into a host cell. It should be noted that the DNA encoding the variable region may also be obtained by performing a total synthesis based on the base sequence determined in section B-2-1 by using an artificial gene synthesis service or the like. Examples of the host cell include animal cells, plant cells, insect cells, yeasts, and bacteria.

B-2-3. Preparation of Monoclonal Antibodies from Host Cell

Monoclonal antibodies may be obtained by culturing the host cell, which has the expression vector incorporated therein, in any appropriate medium, and purifying the monoclonal antibodies from the resultant culture supernatant. A method of purifying antibodies is described above.

B-3. Production of Chimeric Antibodies

Human chimeric antibodies may be obtained by, for example, linking DNA encoding a variable region of a monoclonal antibody obtained in the same manner as above to DNA encoding a constant region of a human antibody, incorporating the resultant into an expression vector, and introducing the expression vector into a host to express human chimeric antibodies (for example, WO 95/14041 A1).

B-4. Production of Humanized Antibodies

Humanized antibodies may be obtained by grafting CDRs of an antibody of a non-human mammal into a human antibody so that the CDRs are linked to the framework region of the human antibody through the use of so-called CDR grafting techniques. A method of grafting CDRs of an antibody of a non-human mammal (such as a mouse) into a human framework region is known, and an example thereof is an overlap extension PCR method.

In general, in CDR grafting, it is advantageous in maintaining the function of the CDRs to select a human framework region having a high homology to the framework region of the antibody of the non-human mammal. Accordingly, it is preferred to utilize a human framework region having an amino acid sequence having a high homology to the amino acid sequence of the framework region adjacent to the CDRs to be grafted.

C. Antibody Fragments

The present invention also provides antibody fragments that include part of a monoclonal antibody described in section A, the antibody fragments being capable of binding to tissue factor. Antibody fragments of the present invention typically have the ability to be internalized by a cell expressing tissue factor. Examples of the antibody fragments include Fab, F(ab′)2, Fab′, a single-chain antibody (scFv), a disulfide-stabilized antibody (dsFv), a dimerized V region fragment (diabody), and a CDR-containing peptide.

The Fab may be obtained by subjecting the monoclonal antibody to papain treatment. In addition, the Fab may also be obtained by inserting DNA encoding the Fab of the monoclonal antibody into any appropriate expression vector, and introducing the vector into a host cell to express the Fab.

The F(ab′)2 may be obtained by subjecting the monoclonal antibody to pepsin treatment. In addition, the F(ab′)2 may also be obtained by inserting DNA encoding the F(ab′)2 of the monoclonal antibody into any appropriate expression vector, and introducing the vector into a host cell to express the F(ab′)2.

The Fab′ is an antibody fragment obtained by cleaving the S—S bond between the hinges of F(ab′)2. The Fab′ may be obtained by subjecting the F(ab′)2 to treatment with the reducing agent dithiothreitol. In addition, the Fab′ may also be obtained by inserting DNA encoding the Fab′ into any appropriate expression vector, and introducing the vector into a host cell to express the Fab′.

The scFv is such that only the variable regions of a heavy chain and a light chain are linked via an appropriate peptide linker. The scFv may be obtained by constructing an expression vector for the scFv based on the DNA encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody, and introducing the vector into a host cell to express the scFv.

The dsFv is such that polypeptides obtained by substituting one amino acid residue in each of a heavy chain variable region and a light chain variable region with a cysteine residue are bound via a S—S bond. The location at which the cysteine residue is introduced in each region may be determined based on a three-dimensional structure predicted by molecular modeling. The dsFv may be obtained by constructing an expression vector for the dsFv based on the DNA encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody, and introducing the vector into a host cell to express the dsFv.

The diabody is a dimer of scFvs linked via a short peptide linker having eight or less amino acid residues, and has divalent antigen binding activity. The divalent antigen binding activity may be identical or different from each other. The diabody may be obtained by constructing an expression vector for scFvs linked via a peptide linker having eight or less amino acid residues based on the DNA encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody, and introducing the vector into a host cell to express the diabody.

The CDR-containing peptide contains at least one of the CDRs of a heavy chain variable region or a light chain variable region. The CDR-containing peptide may be such that a plurality of CDRs are bound directly or via an appropriate peptide linker. The CDR-containing peptide may be obtained by inserting DNA encoding the CDRs in the heavy chain variable region and the light chain variable region of the monoclonal antibody into any appropriate expression vector, and introducing the vector into a host cell to express the CDR-containing peptide. In addition, the CDR-containing peptide may also be obtained by a chemical synthesis method, such as an Fmoc method or a tBoc method.

D. Pharmaceutical Composition

Pharmaceutical compositions of the present invention include: a monoclonal antibody described in section A or an antibody fragment described in section C as a target-binding factor; and a drug. Utilization of a monoclonal antibody or an antibody fragment (hereinafter sometimes referred to as “monoclonal antibody or the like”) as the target-binding factor allows a drug to be efficiently delivered into a cell expressing TF at its surface.

In a first embodiment, the monoclonal antibody or the like may be in a state of being bound to the drug. In addition, in such embodiment, as necessary, a polymer compound may be further bound to the monoclonal antibody or the like.

Any appropriate drug may be selected as the drug depending on the disease to be treated and the like. Examples thereof include: biologics, such as nucleic acid pharmaceuticals, antibody pharmaceuticals, and gene therapy drugs; and cytotoxic molecules, such as cytotoxins and cytotoxic drugs.

Examples of the nucleic acid pharmaceuticals include plasmid DNA, siRNA, micro RNA, shRNA, an antisense nucleic acid, a decoy nucleic acid, an aptamer, and a ribozyme site.

Examples of the cytotoxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, duocarmycin, calicheamicin, maytansine, auristatin, and derivatives thereof.

Examples of the cytotoxic drugs include: metabolic antagonists, suchasmethotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and decarbazine; alkylating agents, such as mechlorethamine, Thio-TEPA, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiaminoplatinum(II); antibiotics, such as anthracyclines including daunorubicin and doxorubicin, dactinomycin, bleomycin, mithramycin, and anthramycin (AMC); and antimitotic agents, such as vincristine and vinblastine.

The binding between the drug or the polymer compound and the monoclonal antibody or the like may be performed by a method known in the art. The binding may be performed by, for example, allowing respective functional groups thereof or functional groups introduced as necessary to react with each other. As a combination of the functional groups, there are given, for example, an amino group and a carboxyl group, a carboxyl group and a hydroxyl group, a maleimide group and a thiol group, a thiol group and a thiol group, a hydrazide group and a ketone group, a hydrazide group and an aldehyde group, an amino group and an aldehyde group, a thiol group and a carboxyl group, an amino group and a squaric acid derivative, a dienyl aldehyde group and an amino group, a halo ester and a thiol group, and an azide and an alkyne. In addition, for example, when the drug is a protein or a peptide, the pharmaceutical composition may contain a fusion protein of the drug and the monoclonal antibody or the like which can be obtained by genetic engineering techniques. Further, when the drug has a charge, the drug may also be bound via an ionic bond.

Any appropriate polymer compound may be selected as the polymer compound. Specific examples thereof include polyethylene glycol, albumin, dextran, polyvinylpyrrolidone, and polyvinyl alcohol. When any such polymer compound is bound, stability in blood can be improved.

The binding site of the polymer compound may be any appropriate site as long as the antigen binding activity and ability to be internalized of the monoclonal antibody or the like are not impaired.

In a second embodiment, the monoclonal antibody or the like may be in a state of being bound to a DDS carrier material. As the DDS carrier material, one capable of forming drug-encapsulated nanoparticles (for example, particles having an average particle diameter of preferably from 10 nm to 400 nm, more preferably from 20 nm to 300 nm, still more preferably from 30 nm to 150 nm) may be preferably used. With the use of such a DDS carrier material, drug-encapsulated nanoparticles can be formed, and hence the in vivo stability of the drug can be improved, and besides, sustained release thereof can be achieved. Further, reliable drug delivery into a target cell by the monoclonal antibody or the like can be achieved.

The monoclonal antibody or the like may be bound at any appropriate location in the DDS carrier material. From the viewpoint of enabling target binding properties to be suitably exhibited, it is preferred that the monoclonal antibody or the like be bound so as to be exposed at the outer surfaces of the nanoparticles formed by the DDS carrier material. The binding between the monoclonal antibody or the like and the DDS carrier material may be performed by, for example, allowing respective functional groups thereof or functional groups introduced as necessary to react with each other. A preferred combination of the functional groups is as described above.

Examples of the DDS carrier material include: polymer micelle-forming materials, such as a block copolymer having a hydrophilic segment and a hydrophobic segment; liposome-forming materials, such as a phospholipid; nanohydrogel capsule-forming materials, such as natural polymers, such as gelatin, collagen, hyaluronic acid, and alginic acid, and synthetic polymers, such as polyethylene glycol and polyvinyl alcohol; and nanosphere-forming materials, such as polyglycolic acid, polylactic acid, and copolymers thereof. Of those, a block copolymer having a hydrophilic segment and a hydrophobic segment is preferably used. When the monoclonal antibody or the like is bound to the hydrophilic segment of the block copolymer having a hydrophilic segment and a hydrophobic segment to prepare a block copolymer having a target binding property, a polymer micelle capable of delivering the drug to a target cell with high reliability can be obtained.

As the drug, a drug similar to that in the first embodiment may be used. In the second embodiment, the drug may be used as is, or may be used in the form of a drug-bound block copolymer by being bound to the hydrophobic segment of the block copolymer having a hydrophilic segment and a hydrophobic segment.

E. Compositions for Drug Delivery

Compositions for drug delivery of the present invention include a monoclonal antibody described in section A or an antibody fragment described in section C. The monoclonal antibody or the antibody fragment can be utilized as a target-binding factor for a cell expressing TF, and can exhibit the ability to be internalized by the cell. Accordingly, when a drug is administered using a composition for drug delivery of the present invention, the drug can be efficiently delivered into a cell expressing TF at its surface.

F. Applications of Anti-TF Monoclonal Antibodies of the Present Invention

Anti-TF monoclonal antibodies of the present invention are typically used for treatment of a disease associated with tissue factor (such as a disease involving enhanced expression of tissue factor at a tissue or cell surface). Examples of diseases associated with tissue factor include cancer, inflammation, and thrombosis. The expression of TF is constitutively enhanced in various cancer tissues, and hence anti-TF monoclonal antibodies can be used for treatment of cancer irrespective of its kind. In addition, for example with respect to pancreatic cancer, the prognosis of patients who highly express TF has been reported to be poor, and it may be effectively utilized also in treatments targeting such patients.

The administration route of the pharmaceutical composition of the present invention is preferably parenteral administration, such as subcutaneous, intravenous, intra-arterial, or local administration, particularly preferably intravenous injection. The dose may be appropriately determined depending on, for example, the kind of the drug, the dosage regimen, the age and gender of the patient, and the patient's state of health.

EXAMPLES

In the following, the present invention is described in more detail by way of Examples. However, the present invention is by no means limited to these Examples. It should be noted that the term “part(s)” and the term “%” mean “part(s) by weight” and “wt %”, respectively.

Example 1 Anti-hTF Monoclonal Antibodies and Fragments Thereof [Preparation of Antigen]

A recombinant protein containing an amino acid sequence from position 33 to position 251 in the full-length amino acid sequence of hTF was expressed using Escherichia coli, and purified with a nickel column to afford recombinant hTF (SEQ ID NO: 29), which was used as the antigen.

[Immunization of Rats]

50 μg of the recombinant hTF was intraperitoneally coadministered with Freund's complete adjuvant (Difco) to three 6-week-old Wistar female rats and thus the initial immunization was performed. After 14 days therefrom, 50 μg of the recombinant hTF was coadministered with Sigma Adjuvant System (Sigma) and thus a booster immunization was performed. Thereafter, similar booster immunizations were performed every 21 days five times. After an additional 126 days, 10 μg of the recombinant hTF diluted with PBS was intraperitoneally administered and 40 μg of the recombinant hTF was administered to the tail vein; thus, the final immunization was performed.

[Preparation of Hybridomas]

After 3 days from the final immunization, the spleen was excised, and spleen cells were collected. The spleen cells and mouse myeloma cells (p3X63Ag8.653) were fused using polyethylene glycol 4000 (Merck) at a concentration of 50%, and selection was performed with HAT medium.

[Screening of Antibody-Producing Hybridomas]

After 8 days from the cell fusion, screening of antibody-producing hybridomas was performed. The immunoassay used for the screening is the following. For an ELISA method, a 50 mM carbonate buffer (pH 8) containing 1 μg/mL of the recombinant hTF was added to each well of a 96-well microtiter plate (manufactured by Nunc) at 50 μL/well, and immobilization was performed at 4° C. overnight or at room temperature for 2 hours. The wells were washed three times with 300 μL of a washing solution (0.05% Tween 20/25 mM Tris/140 mM NaCl/2.5 mM KCl, pH 7.4), and then 200 μL of a blocking buffer (0.05% Tween 20/1% BSA/100 mM NaH2PO4/140 mM NaCl, pH 5) was added, followed by standing at 4° C. overnight or at room temperature for 1 hour to undergo blocking. 50 μL of a hybridoma culture supernatant was added to each well of the thus-obtained hTF-immobilized plate and the mixture was allowed to react at room temperature for 1 hour. Each well was washed three times with 300 μL of the washing solution. After that, 50 μL of an HRP-labeled anti-mouse IgG antibody (Bethyl) diluted 5,000-fold with the blocking buffer was added, and the mixture was allowed to react for 30 minutes. After the reaction, each well was washed three times with 300 μL of the washing solution, and 100 μL of 3.7 mM o-phenylenediamine/25 mM citric acid/130 mM Na2HPO4/0.006% H2O2 (pH 5.0) was added to develop color. After from 10 minutes to 15 minutes, 2 N sulfuric acid was added at 30 μL/well to stop the reaction, and absorbance (490 nM) was measured with an absorbance plate reader. In addition, an immunoprecipitation ELISA method was performed by mixing the recombinant hTF and the hybridoma culture supernatant, and measuring the amount of unbound hTF in the mixed liquid using an hTF-quantifying sandwich ELISA. Further, a flow cytometry method was performed in accordance with a conventional method to measure the reactivity of the hybridoma culture supernatant to hTF-expressing cells.

Hybridomas that showed a strong affinity for hTF as a result of the measurement were selected, and a limiting dilution method was performed twice for clones of the hybridomas to establish hybridoma clones producing monoclonal antibodies that bind to hTF.

[Preparation of Antibodies]

Each established hybridoma was mass-cultured in, for example, RPMI 1640 medium containing bovine serum in an amount of 5% having removed therefrom IgG of bovine origin, to afford a culture supernatant. Alternatively, each hybridoma was mass-cultured in the abdominal cavity of ICR nude mice, and peritoneal fluid was collected. The resultant culture supernatant or peritoneal fluid was subjected to Protein G affinity column chromatography to purify IgG monoclonal antibodies.

[Internalization Assay]

Each monoclonal antibody obtained as described above was subjected to the following internalization assay.

TF-high-expressing human pancreatic cancer cells BxPC3 were seeded into a four-chamber Culture Slide (BD) at 5×104 cells/chamber, and cultured in RPMI 1640 medium at 37° C. under a 5% CO2 environment for 12 hours. The resultant was washed three times with PBS, and then 30 μg of antibody labeled with an Alexa 647 fluorescence labeling kit (Invitrogen) was diluted with 1 ml of RPMI 1640 medium and added to each chamber, followed by culturing for 3 hours. In the 3-hour culture, after a lapse of 2 hours from the initiation of the culture, Lysotracker RED-DND99 (Invitrogen) was added to the culture solution to have a final concentration of 75 nM, followed by further culturing for 1 hour. Then, the resultant was washed three times with PBS. After that, immobilization was performed with 4% paraformaldehyde, and nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI), followed by mounting with Fluoromount-G (Southern Biotech). Then, the cells were observed using a fluorescence microscope (Keyence). The results of the observation are shown in FIG. 1.

As shown in FIG. 1(a) and FIG. 1(b), in two of the monoclonal antibodies (No. 1849 and No. 1859), the labeling substance transferred into cells, and thus it was confirmed that these monoclonal antibodies had the ability to be internalized. On the other hand, the ability to be internalized was not confirmed for the antibody shown in FIG. 1(c). It should be noted that the subclasses of those antibodies were determined using Mouse Monoclonal Antibody Isotyping ELISA Kit (manufactured by BD Biosciences) and the results were as follows: the subclass of No. 1849 was IgG2b and the subclass of No. 1859 was IgG2a.

[Determination of DNA Sequences Encoding Variable Regions, Amino Acid Sequences, and CDR Sequences]

1. Anti-hTF Monoclonal Antibody (No. 1849)

Total RNA was extracted from hybridomas producing the anti-hTF monoclonal antibody (No. 1849), and then cDNA was synthesized from the resultant total RNA, in accordance with a conventional method.

DNA fragments encoding the heavy chain variable region and the light chain variable region of the anti-hTF monoclonal antibody (No. 1849) were obtained by a PCR method using the synthesized cDNA as a template. Specifically, PCR was performed using, from the following list of primers, a mixed primer of the following primers 1 to 19 and the following primer 20 as a sense primer and antisense primer for heavy chain variable region cloning, respectively, and a mixed primer of the following primers 22 to 38 and the following primer 39 as a sense primer and antisense primer for light chain variable region cloning, respectively.

[List of Primers] Primer 1 (SEQ ID NO: 30) NNCCATGGCCGAGGTRMAGCTTCAGGAGTC Primer 2 (SEQ ID NO: 31) NNCCATGGCCGAGGTBCAGCTBCAGCAGTC Primer 3 (SEQ ID NO: 32) NNCCATGGCCGAGGTGCAGCTGAAGSASTC Primer 4 (SEQ ID NO: 33) NNCCATGGCCGAGGTCCARCTGCAACARTC Primer 5 (SEQ ID NO: 34) NNCCATGGCCGAGGTYCAGCTBCAGCARTC Primer 6 (SEQ ID NO: 35) NNCCATGGCCGAGGTYCARCTGCAGCAGTC Primer 7 (SEQ ID NO: 36) NNCCATGGCCGAGGTCCACGTGAAGCAGTC Primer 8 (SEQ ID NO: 37) NNCCATGGCCGAGGTGAASSTGGTGGAATC Primer 9 (SEQ ID NO: 38) NNCCATGGCCGAGGTGAWGYTGGTGGAGTC Primer 10 (SEQ ID NO: 39) NNCCATGGCCGAGGTGCAGSKGGTGGAGTC Primer 11 (SEQ ID NO: 40) NNCCATGGCCGAGGTGCAMCTGGTGGAGTC Primer 12 (SEQ ID NO: 41) NNCCATGGCCGAGGTGAAGCTGATGGARTC Primer 13 (SEQ ID NO: 42) NNCCATGGCCGAGGTGCARCTTGTTGAGTC Primer 14 (SEQ ID NO: 43) NNCCATGGCCGAGGTRAAGCTTCTCGAGTC Primer 15 (SEQ ID NO: 44) NNCCATGGCCGAGGTGAARSTTGAGGAGTC Primer 16 (SEQ ID NO: 45) NNCCATGGCCGAGGTTACTCTRAAAGWGTSTG Primer 17 (SEQ ID NO: 46) NNCCATGGCCGAGGTCCAACTVCAGCARCC Primer 18 (SEQ ID NO: 47) NNCCATGGCCGAGGTGAACTTGGAAGTGTC Primer 19 (SEQ ID NO: 48) NNCCATGGCCGAGGTGAAGGTCATCGAGTC Primer 20 (SEQ ID NO: 49) TGTGCAGACCCTCGTGGACCACGGAGCA Primer 21 (SEQ ID NO: 50) GGACTCTGGGRTCATTTACCMGGAGAGT Primer 22 (SEQ ID NO: 51) NNNNGTCGACGCTCGAYATCCAGCTGACTCAGCC Primer 23 (SEQ ID NO: 52) NNNNGTCGACGCTCGAYATTGTTCTCWCCCAGTC Primer 24 (SEQ ID NO: 53) NNNNGTCGACGCTCGAYATTGTGMTMACTCAGTC Primer 25 (SEQ ID NO: 54) NNNNGTCGACGCTCGAYATTGTGYTRACACAGTC Primer 26 (SEQ ID NO: 55) NNNNGTCGACGCTCGAYATTGTRATGACMCAGTC Primer 27 (SEQ ID NO: 56) NNNNGTCGACGCTCGAYATTMAGATRAMCCAGTC Primer 28 (SEQ ID NO: 57) NNNNGTCGACGCTCGAYATTCAGATGAYDCAGTC Primer 29 (SEQ ID NO: 58) NNNNGTCGACGCTCGAYATYCAGATGACACAGAC Primer 30 (SEQ ID NO: 59) NNNNGTCGACGCTCGAYATTGTTCTCAWCCAGTC Primer 31 (SEQ ID NO: 60) NNNNGTCGACGCTCGAYATTGWGCTSACCCAATC Primer 32 (SEQ ID NO: 61) NNNNGTCGACGCTCGAYATTSTRATGACCCARTC Primer 33 (SEQ ID NO: 62) NNNNGTCGACGCTCGAYRTTKTGATGACCCARAC Primer 34 (SEQ ID NO: 63) NNNNGTCGACGCTCGAYATTGTGATGACBCAGKC Primer 35 (SEQ ID NO: 64) NNNNGTCGACGCTCGAYATTGTGATAACYCAGGA Primer 36 (SEQ ID NO: 65) NNNNGTCGACGCTCGAYATTGTGATGACCCAGWT Primer 37 (SEQ ID NO: 66) NNNNGTCGACGCTCGAYATTGTGATGACACAACC Primer 38 (SEQ ID NO: 67) NNNNGTCGACGCTCGAYATTTTGCTGACTCAGTC Primer 39 (SEQ ID NO: 68) CCTTAGGAGGGAAGATTGGAAGGAGCT

It should be noted that, in the base sequences, R represents G or A, Y represents T or C, M represents A or C, K represents G or T, S represents G or C, W represents A or T, B represents G, C, or T, D represents A, G, or T, V represents A, G, or C, and N represents A, T, G, or C.

Each PCR product obtained in the foregoing was cloned into a vector to determine its base sequence in accordance with a conventional method.

The base sequences of the heavy chain variable region and the light chain variable region of the anti-hTF monoclonal antibody (No. 1849) determined as described above are set forth in SEQ ID NOS: 69 and 70, respectively. In addition, the amino acid sequences of the heavy chain variable region and the light chain variable region are set forth in SEQ ID NOS: 9 and 10, respectively. In addition, those amino acid sequences were compared to a database of the amino acid sequences of known antibodies (website of IMGT: http://www.imgt.org/) to investigate their homologies. Thus, the amino acid sequences of the CDRs were determined to be as follows.

TABLE 1 No. 1849 Amino acid sequence SEQ ID NO: Heavy chain CDR1 DYNMA 3 variable CDR2 AIIYDGTRTYYRDSVRG 4 region CDR3 GDSYTNFAY 5 Light chain CDR1 RASSSLSYMH 6 variable CDR2 ETSKLAS 7 region CDR3 QQGNSYPRT 8

2. Anti-hTF Monoclonal Antibody (No. 1859)

The base sequences of the heavy chain variable region and the light chain variable region of the anti-hTF monoclonal antibody (No. 1859) were determined in the same manner as above except that total RNA was extracted from hybridomas producing the anti-hTF monoclonal antibody (No. 1859) and primer 21 was used as the antisense primer for the heavy chain variable region cloning.

The determined base sequences of the heavy chain variable region and the light chain variable region of the anti-hTF monoclonal antibody (No. 1859) are set forth in SEQ ID NOS: 71 and 72, respectively. In addition, the amino acid sequences of the heavy chain variable region and the light chain variable region are set forth in SEQ ID NOS: 17 and 18, respectively. In addition, the amino acid sequences of the CDRs in those variable regions were determined to be as follows.

TABLE 2 Amino acid No. 1859 sequence SEQ ID NO: Heavy chain CDR1 DYSVH 11 variable CDR2 VMWSGGTTTFNSGLKS 12 region CDR3 ERAGSPLNWFAY 13 Light chain CDR1 QASQDIGNYLS 14 variable CDR2 SSTSLAD 15 region CDR3 LQHYSGSRT 16

[Binding Activity Evaluation by Surface Plasmon Resonance Method]

The anti-hTF monoclonal antibodies (No. 1849 and No. 1859) were each immobilized on the surface of a Biacore CM5 chip. The CM5 chip, which had each antibody immobilized thereon, was set in a SPR device, and an antigen-containing buffer containing purified hTF was allowed to flow through its flow path. The dissociation constant between each of the antibodies and hTF was determined by this measurement system. It should be noted that the measurement conditions were as follows.

SPR apparatus: Biacore 2000 (Biacore)
Antibody-containing buffer: 10 mM acetate buffer (pH5.0) containing hTF at a final concentration of 25 μg/ml
Running buffer: HBS-EP buffer (10 mM HEPES, pH 7.5, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20 (Tween 20), pH 7.4).

As a result of the measurement, the dissociation constants (KD) between hTF and the antibodies No. 1849 and No. 1859 were 9.139×10−11 and 1.894×10−10, respectively.

[Anticoagulant Activity Evaluation]

The prothrombin time of each of the anti-hTF monoclonal antibodies (No. 1849 and No. 1859) was measured as described below.

3 μg of the antibody was added to 350 ng of a recombinant hTF antigen, and PBS was added to a total volume of 5 μl. An antigen-antibody reaction was performed for 15 minutes under shaking at 37° C. and 600 rpm. To the reaction solution, 50 μl of human serum subjected to anticoagulant treatment with 3.8% sodium citrate, and 100 μl of 25 mM CaCl2 were added, and at the same time, culturing was performed under shaking at 37° C. and 600 rpm. The period of time until a fibrin clot was formed (prothrombin time) was measured. The prolonged coagulation time ratio (prothrombin time ratio) of each antibody when the prothrombin time in a control using PBS in place of the reaction solution is defined as 1 are shown in Table 3 and FIG. 2.

TABLE 3 No. 1849 No. 1859 Control (PBS) Prolonged 5.535 1.222 1 coagulation time ratio

As shown in Table 3 and FIG. 2, the prolonged coagulation time ratio of No. 1859 is 1.222, and thus its anticoagulant activity was found to be extremely small.

Example 2 Pharmaceutical Composition [Binding Between Monoclonal Antibody and Drug]

The anti-hTF monoclonal antibody (No. 1849) having monomethyl auristatin E (MMAE) bound thereto (hereinafter referred to as “hTF-MMAE”) was obtained as described below.

To an aqueous solution (18 mL) of H-Cit-OH (1.18 g, 6.74 mmol) and NaHCO3 (566 mg, 6.74 mmol), a solution of Fmoc-Val-OSu (2.80 g, 6.42 mmol) in DMF (18 mL) was added, and THF (9 mL) was further added. The mixture was stirred overnight. The reaction was stopped with a 15% citric acid aqueous solution (40 mL), and the aqueous layer was extracted with an AcOEt/i-PrOH (9/1) mixed solution (100 mL, 20 mL×2). The combined organic layer was washed with water (70 mL), and concentrated under reduced pressure. The residual solid was washed with diethyl ether to afford a dipeptide (3.11 g, 97%) as a white solid.

To a solution of Fmoc-Val-Cit-OH (3.00 g, 6.04 mmol) and p-aminobenzyl alcohol (1.49 g, 12.1 mmol) in dichloromethane (70 mL) and methanol (30 mL), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (2.99 g, 12.1 mmol) was added. After 1 day, EEDQ (1.50 g, 6.04 mmol) was further added, and the mixture was stirred overnight. The reaction solution was concentrated, and the residue was washed with diethyl ether to afford the target product (2.51 g, 690).

Fmoc-Val-Cit-PAB-OH (2.00 g, 3.32 mmol) was dissolved in dimethylformamide (20 mL), and bis-p-nitrophenyl carbonate (2.02 mg, 6.65 mmol) and diisopropylethylamine (0.87 mL, 4.98 mmol) were added. After stirring overnight, the reaction solution was concentrated under reduced pressure, and the residue was washed with ethyl acetate and diethyl ether to afford a p-nitrophenyl carbonate (1.73 g, 68%).

To a solution of the p-nitrophenyl carbonate (1.28 g, 1.67 mmol) and HOBt (376 mg, 2.78 mmol) in dimethylformamide (3.4 mL) and pyridine (0.85 mL), MMAE (1.00 g, 1.39 mmol) was added. After 24 hours, the reaction solution was purified with Sephadex LH20 (solvent: CHCl3:MeOH=1:1) to afford Fmoc-Val-Cit-PABC-MMAE (1.44 g, 77%).

To a solution (20 mL) of Fmoc-Val-Cit-PABC-MMAE (1.44 g, 1.07 mmol) in dimethylformamide, Et2NH (5 mL) was added. After stirring overnight, the reaction solution was concentrated under reduced pressure, and the residue was washed with ethyl acetate and diethyl ether to afford a pale yellow solid (960 mg, 800).

To a solution (20 mL) of H-Val-Cit-PABC-MMAE (960 mg, 0.855 mmol) in dichloromethane, Mal-PEG12-OSu having a maleimide group at the N-terminus (814 mg, 0.94 mmol) and diisopropylethylamine (0.45 mmol, 2.57 mmol) were added. After stirring overnight, the reaction solution was purified through the use of Sephadex LH20 (CHCl3:MeOH=1:1) and gel filtration HPLC to afford Mal-PEG12-Val-Cit-PABC-MMAE (hereinafter sometimes referred to as “maleimide MMAE compound”) as a colorless oil (769 mg, 48%).

A buffer having a pH of 6.4 was prepared using 5 mM EDTA-containing PBS, and a 150 mM NaCl and 5 mM EDTA-containing 100 mM phosphate buffer (pH 6.0), and an antibody solution was prepared with the resultant buffer to have an antibody concentration of 1.0 mg/ml. To the antibody solution, dithiothreitol (DTT) was added to have a final concentration of from 1 mM to 10 mM, and the mixture was allowed to react at from 26° C. to 37° C. for from 30 minutes to 45 minutes. Then, Amicon Ultra (MWCO: 30,000) was used to remove the reaction reagent from the reaction solution. Absorption was measured, and the recovery ratio of the antibody was found to be from 80% to 99%. In addition, the result of quantification of SH groups based on 5,5′-dithiobis(2-nitrobenzoic acid) (DNTB) revealed that three to five SH groups were obtained per antibody.

Next, a buffer having a pH of 6.4 was prepared using 5 mM EDTA-containing PBS, and a 150 mM NaCl and 5 mM EDTA-containing 100 mM phosphate buffer (pH 6.0), and the reaction solution was diluted with the resultant buffer to have a protein concentration of 0.5 mg/ml. The diluted solution was mixed with the maleimide MMAE compound at a molar ratio of 1:4 (antibody:maleimide MMAE compound), and the resultant was allowed to react at room temperature for 1 hour and then at 4° C. overnight.

After that, Amicon Ultra (MWCO: 30,000) was used to remove the reaction reagent from the reaction solution, and then the solvent was replaced with PBS. A protein was recovered with Amicon Ultra, and the recovery ratio was from 60% to 90%.

Thus, hTF-MMAE having three to five MMAE molecules added per antibody was obtained.

[Cytocidal Effect Confirmation Test]

Human pancreatic cancer cells (BxPC3) were added to a 96-well plate at 3×103 cells/well, and cultured in RPMI medium containing 10% FCS, penicillin (100 U/ml), and streptomycin (100 μg/ml) hTF-MMAE was added to the wells at various drug concentrations, and cell survival rates 72 hours after the addition were calculated.

It should be noted that, as a comparative test, a similar test was performed using, in place of hTF-MMAE, mTF-MMAE obtained by binding MMAE, in the same manner as above, to a monoclonal antibody (mTF) against mouse TF not expressed in human cancer cells, and a cell survival rate was calculated. The ratio (%) of each cell survival rate, with the cell survival rate of a negative control (no addition of a drug sample) being defined as 100%, is shown in FIG. 3.

As shown in FIG. 3, hTF-MMAE showed a remarkably excellent cytocidal effect as compared to mTF-MMAE. This indicates that, when a drug is bound to the anti-hTF monoclonal antibody (No. 1849), its property of being transferred into a cell expressing hTF at its surface can be improved to allow high efficacy of the drug to be exhibited.

[Antitumor Effect Confirmation Test]

1×107 cells/100 μL of human pancreatic cancer cells (BxPC3) were implanted subcutaneously into the backs of 4-week-old nude mice (BALBc nu/nu, female), and treatment was initiated when the tumor volume (calculated based on the tumor diameter) reached about 200 mm3. The day of initiation of the treatment was defined as Day 0, and a drug was administered to the tail vein once on each of Day 0, Day 4, and Day 8 (administration three times in total). hTF-MMAE was used as the drug, and physiological saline was administered as a control (each group: N=7). The dose of the drug was 10 mg/kg per administration (antibody amount: about 200 μg/mouse). Thereafter, the tumor diameter and the body weight were measured twice a week until Day 30. The ratios of the tumor volume and the body weight to the tumor volume and the body weight at Day 0 are shown in FIG. 4 and FIG. 5, respectively.

As shown in FIG. 4, hTF-MMAE showed a remarkably excellent antitumor effect. The result agrees with the cytocidal effect confirmed in vitro. In addition, as shown in FIG. 5, a reduction in body weight was not found in the group to which hTF-MMAE had been administered.

Example 3 Anti-mTF Monoclonal Antibody and Fragment Thereof [Preparation of Antigen]

A recombinant protein containing an amino acid sequence from position 30 to position 251 in the full-length amino acid sequence of mTF was expressed using Escherichia coli, and purified with a nickel column to afford recombinant mTF (SEQ ID NO: 73), which was used as the antigen.

[Immunization of Rats]

50 μg of the recombinant mTF was intraperitoneally coadministered with Freund's complete adjuvant (Difco) to three 6-week-old Wistar female rats and thus initial immunization was performed. After 14 days therefrom, 50 μg of the recombinant mTF was coadministered with Sigma Adjuvant System (Sigma) and thus a booster immunization was performed. Thereafter, similar booster immunizations were performed every 21 days seven times. After an additional 207 days, 10 μg of the recombinant mTF diluted with PBS was intraperitoneally administered and 40 μg of the recombinant mTF was administered to the tail vein; thus, final immunization was performed.

[Preparation of Hybridomas]

After 3 days from the final immunization, the spleen was excised, and spleen cells were collected. The spleen cells and mouse myeloma cells (p3X63Ag8.653) were fused using polyethylene glycol 4000 (Merck) at a concentration of 50%, and selection was performed in HAT medium.

[Screening of Antibody-Producing Hybridomas]

After 8 days from the cell fusion, screening of antibody-producing hybridomas was performed. The immunoassay used for the screening is the following. For an ELISA method, a 50 mM carbonate buffer (pH 8) containing 1 μg/mL of the recombinant mTF was added to each well of a 96-well microtiter plate (manufactured by Nunc) at 50 μL/well, and immobilization was performed at 4° C. overnight or at room temperature for 2 hours. The wells were washed three times with 300 μL of a washing solution (0.05% Tween 20/25 mM Tris/140 mM NaCl/2.5 mM KCl, pH 7.4), and then 200 μL of a blocking buffer (0.05% Tween 20/1% BSA/100 mM NaH2PO4/140 mM NaCl, pH 5) was added, followed by standing at 4° C. overnight or at room temperature for 1 hour to undergo blocking. 50 μL of a hybridoma culture supernatant was added to each well of the thus-obtained mTF-immobilized plate and the mixture was allowed to react at room temperature for 1 hour. Each well was washed three times with 300 μL of the washing solution, and then 50 μL of an HRP-labeled anti-mouse IgG antibody (Bethyl) diluted 5,000-fold with the blocking buffer was added, and the mixture was allowed to react for 30 minutes. After the reaction, each well was washed three times with 300 μL of the washing solution, and 100 μL of 3.7 mM o-phenylenediamine/25 mM citric acid/130 mM Na2HPO4/0.006% H2O2 (pH 5.0) was added to develop a color. After from 10 minutes to 15 minutes, 2 N sulfuric acid was added at 30 μL/well to stop the reaction, and absorbance (490 nM) was measured with an absorbance plate reader. In addition, an immunoprecipitation ELISA method was performed by mixing the recombinant mTF and the hybridoma culture supernatant, and measuring the amount of unbound mTF in the mixed liquid using an mTF-quantifying sandwich ELISA. Further, a flow cytometry method was performed in accordance with a conventional method to measure the reactivity of the hybridoma culture supernatant to mTF-expressing cells.

Hybridomas that showed a strong affinity for mTF as a result of the measurement were selected, and a limiting dilution method was performed twice for clones of the hybridomas to establish hybridoma clones producing monoclonal antibodies that bind to mTF.

[Preparation of Antibodies]

Each established hybridoma was mass-cultured in, for example, RPMI 1640 medium containing bovine serum in an amount of 5% having removed therefrom IgG of bovine origin, to afford a culture supernatant. Alternatively, each hybridoma was mass-cultured in the abdominal cavity of ICR nude mice, and peritoneal fluid was collected. The resultant culture supernatant or peritoneal fluid was subjected to Protein G affinity column chromatography to purify IgG monoclonal antibodies.

[Internalization Assay]

Each monoclonal antibody obtained as described above was subjected to the following internalization assay.

Mouse B16 melanoma cells and TF-forced-expression cells thereof were seeded into a four-chamber Culture Slide (BD) at 5×104 cells/chamber, and cultured in RPMI 1640 medium at 37° C. under a 5% CO2 environment for 12 hours. The resultant was washed three times with PBS, and then 30 μg of antibody labeled with an Alexa 647 fluorescence labeling kit (Invitrogen) was diluted with 1 ml of RPMI 1640 medium and added to each chamber, followed by culturing for 3 hours. In the 3-hour culture, after a lapse of 2 hours from the initiation of the culture, Lysotracker RED-DND99 (Invitrogen) was added to the culture solution to have a final concentration of 75 nM, followed by further culturing for 1 hour. Then, the resultant was washed three times with PBS. After that, immobilization was performed with 4% paraformaldehyde, and nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI), followed by mounting with Fluoromount-G (Southern Biotech). Then, the cells were observed using a fluorescence microscope (Keyence). The result of the observation is shown in FIG. 6.

As shown in FIG. 6, in one monoclonal antibody (No. 1157), the fluorescence-labeled antibody (red) transferred into cells, and thus it was confirmed that the monoclonal antibody had the ability to be internalized by a cell expressing mTF. It should be noted that, in FIG. 6, cells into which the fluorescence-labeled antibody (red) transferred were presumed to be the TF-forced-expression mouse B16 melanoma cells and cells into which the antibody did not transfer were presumed to be the normal mouse B16 melanoma cells (for reference, the enhancement level of the TF expression amount in the TF-forced-expression mouse B16 melanoma cells is shown in FIG. 7. FIG. 7 is a graph that shows the ratios of the mRNA expression amounts of TF to the mRNA expression amounts of GAPDH in the mouse B16 melanoma cells and the TF-forced-expression cells thereof).

[Determination of DNA Sequence Encoding Variable Regions, Amino Acid Sequences, and CDR Sequences]

The base sequences of the heavy chain variable region and the light chain variable region of the anti-mTF monoclonal antibody (No. 1157) were determined in the same manner as in the method of determining the base sequences of the heavy chain variable region and the light chain variable region of the anti-hTF monoclonal antibody (No. 1849) except that total RNA was extracted from hybridomas producing the anti-mTF monoclonal antibody (No. 1157).

The determined base sequences of the heavy chain variable region and the light chain variable region of the anti-mTF monoclonal antibody (No. 1157) are set forth in SEQ ID NOS: 74 and 75, respectively. In addition, the amino acid sequences of the heavy chain variable region and the light chain variable region are set forth in SEQ ID NOS: 25 and 26, respectively. In addition, the amino acid sequences of the CDRs in those variable regions were determined to be as follows.

TABLE 4 Amino acid No. 1157 sequence SEQ ID NO: Heavy chain CDR1 TDYGM 19 variable CDR2 SITVRNYIYYADTVK 20 region CDR3 RTEGMDY 21 Light chain CDR1 KVSQNINGYLN 22 variable CDR2 NTDNLQT 23 region CDR3 LQHYSWPLT 24

Example 4 Production of Chimeric Antibodies

DNA fragments encoding the heavy chain variable region and the light chain variable region of the anti-hTF monoclonal antibody (No. 1849) cloned in Example 1 were amplified by PCR. The DNA fragment for the heavy chain variable region was inserted into a human IgG1 heavy chain constant region-expressing cloning vector (pFUSEss_CHIg-hG1e2 (invivoGen)) and the DNA fragment for the light chain variable region was inserted into a human kappa light chain constant region-expressing cloning vector (pFUSE2ss_CLIg_hk (invivoGen)) to afford expression vectors. The resultant expression vectors were transfected into CHO-K1 cells using Lipofectamine LTX Reagent (Invitrogen). Then, drug selection was performed with 10 μg/mL of Blastcidin S (Kaken Pharmaceutical Co., Ltd.) and 300 μg/mL of Zeocin (Invitrogen) to afford a double-resistant cell line.

The resultant cell line was maintained and cultured in a medium containing Ham's F12K (Wako), 10% FBS, 1% penicillin, streptomycin (Invitrogen), 10 μg/mL of Blastcidin S, and 300 μg/mL of Zeocin. Then, a constitutively anti-hTF human chimeric antibody-expressing cell line (No. 1849 chimeric clone) was cloned by a limiting dilution method that involved using a 96-well plate.

The culture supernatant of the cloned cell line (No. 1849 chimeric clone) was subjected to ELISA, and as a result, reactivity to hTF was able to be confirmed. In addition, the culture supernatant of the cloned cell line was subjected to flow cytometry analysis (FACS), and as a result, reactivity to human colon adenocarcinoma cells (DLD-1) was able to be confirmed. Specifically, as shown in FIG. 8, the culture supernatant showed specific reactivity to hTF-expressing cells DLD-1 (in FIG. 8, (1), (2), and (3) indicate analysis results using the rat anti-hTF monoclonal antibody (No. 1849), the culture supernatant of the No. 1849 chimeric clone, and a rat isotype control, respectively).

[Determination of DNA Sequences Encoding Chimeric Antibody and Amino Acid Sequences]

A vector was extracted from the No. 1849 chimeric clone, and the base sequences and the amino acid sequences of the heavy chain and the light chain of the anti-hTF human chimeric antibody (No. 1849) encoded by the vector were determined in accordance with a conventional method. The determined base sequences of the heavy chain and the light chain of the chimeric antibody are set forth in SEQ ID NOS: 76 and 77, respectively. In addition, the determined amino acid sequences of the heavy chain and the light chain of the chimeric antibody are set forth in SEQ ID NOS: 78 and 79, respectively.

INDUSTRIAL APPLICABILITY

Monoclonal antibodies or fragments thereof of the present invention can be suitably utilized in the field of DDS.

Claims

1. A monoclonal antibody or biologically active fragment thereof, which binds to tissue factor, selected from the group consisting of:

an anti-human tissue factor monoclonal antibody or biologically active fragment thereof including a heavy chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 3, 4, and 5, respectively, and a light chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in represented by SEQ ID NOS: 6, 7, and 8, respectively;
an anti-human tissue factor monoclonal antibody or biologically active fragment thereof including a heavy chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in represented by SEQ ID NOS: 11, 12, and 13, respectively, and a light chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in represented by SEQ ID NOS: 14, 15, and 16, respectively; and
an anti-mouse tissue factor monoclonal antibody or biologically active fragment thereof including a heavy chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 19, 20, and 21, respectively, and a light chain variable region having complementarity determining regions 1, 2, and 3 containing the amino acid sequences set forth in SEQ ID NOS: 22, 23, and 24, respectively.

2. The monoclonal antibody according to claim 1, selected from the group consisting of:

an anti-human tissue factor monoclonal antibody or biologically active fragment thereof including a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence which is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence which is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 10;
an anti-human tissue factor monoclonal antibody or biologically active fragment thereof including a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence which is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence which is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 18; and
an anti-mouse tissue factor monoclonal antibody or biologically active fragment thereof including a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence which is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence which is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 26.

3.-4. (canceled)

5. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a human chimeric antibody or a humanized antibody.

6.-7. (canceled)

8. A pharmaceutical composition, comprising:

the monoclonal antibody or biologically active fragment thereof of claim 1 as a target-binding factor; and
a drug.

9. A composition for drug delivery, comprising the monoclonal antibody or biologically active fragment thereof of claim 1 as a target-binding factor.

10. A pharmaceutical composition, comprising:

the monoclonal antibody or biologically active fragment thereof of claim 2 as a target-binding factor; and
a drug.

11. A composition for drug delivery, comprising the monoclonal antibody or biologically active fragment thereof of claim 2 as a target-binding factor.

12. An isolated nucleic acid sequence encoding an antibody or biologically active fragment thereof comprising the heavy chain CDR sequences and light chain CDR sequences according to claim 1.

13. An isolated nucleic acid sequence encoding an antibody or antigen-binding fragment thereof according to claim 2.

14. An expression vector comprising the nucleic acid sequence of claim 12.

15. An expression vector comprising the nucleic acid sequence of claim 13.

16. A host cell comprising the vector of claim 14.

17. A host cell comprising the vector of claim 15.

18. A method for treating cancer, inflammation, or thrombosis, comprising administering to a patient in need thereof the monoclonal antibody or biologically active fragment thereof according to claim 1.

19. A method for treating cancer, inflammation, or thrombosis, comprising administering to a patient in need thereof the monoclonal antibody or biologically active fragment thereof according to claim 2.

20. A method for treating cancer, inflammation, or thrombosis, comprising administering to a patient in need thereof the pharmaceutical composition according to claim 8.

21. A method for treating cancer, inflammation, or thrombosis, comprising administering to a patient in need thereof the pharmaceutical composition according to claim 10.

22. The method for treating cancer, inflammation, or thrombosis according to claim 18, wherein the monoclonal antibody or biologically active fragment thereof binds to tissue factor at a dissociation constant (KD) of 2×10−10 M or less.

23. The monoclonal antibody or biologically active fragment thereof according to claim 1, wherein the monoclonal antibody or biologically active fragment thereof binds to tissue factor at a dissociation constant (KD) of 2×10−10 M or less.

Patent History
Publication number: 20160333113
Type: Application
Filed: Feb 3, 2015
Publication Date: Nov 17, 2016
Patent Grant number: 9920133
Applicants: NATIONAL CANCER CENTER (Tokyo), The University of Tokyo (Tokyo), RIKEN (Wako-shi), NanoCarrier Co., Ltd. (Kashiwa-shi)
Inventors: Yasuhiro MATSUMURA (Kashiwa-shi), Masahiro YASUNAGA (Kashiwa-shi), Yoshikatsu KOGA (Kashiwa-shi), Yoshiyuki YAMAMOTO (Kashiwa-shi), Ryuta SATO (Kashiwa-shi), Ryo TSUMURA (Kashiwa-shi), Kazunori KATAOKA (Tokyo), Nobuhiro NISHIYAMA (Tokyo), Yutaka MIURA (Tokyo), Shino MANABE (Wako-shi), Yasuki KATO (Kashiwa-shi)
Application Number: 15/111,875
Classifications
International Classification: C07K 16/36 (20060101); A61K 47/48 (20060101); A61K 39/395 (20060101);